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Zinc Switch in Pig Heart Lipoamide Dehydrogenase: Steady-State and Transient Kinetic Studies of the Diaphorase Reaction

I. G. Gazaryan1,2,3,4, V. A. Shchedrina3, N. L. Klyachko3,5, A. A. Zakhariants4,6, S. V. Kazakov2, and A. M. Brown1,a*

1Department of Cell Biology and Anatomy, New York Medical College, 15 Dana Road, Valhalla, NY 10605, USA

2Department of Chemistry and Physical Sciences, Dyson College of Arts and Sciences, Pace University, 861 Bedford Road, Pleasantville, NY 10570

3Department of Chemical Enzymology, Lomonosov Moscow State University, 119899 Moscow, Russia

4Bach Institute of Biochemistry, Federal Research Centre “Fundamentals of Biotechnology”, Russian Academy of Sciences, 119071 Moscow, Russia

5Derzhavin Tambov State University, 392000 Tambov, Russia

6Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Russian Academy of Sciences, 117997 Moscow, Russia

* To whom correspondence should be addressed.

Received May 27, 2020; Revised June 11, 2020; Accepted June 14, 2020
Elevation of intracellular Zn2+ following ischemia contributes to cell death by affecting mitochondrial function. Zn2+ is a differential regulator of the mitochondrial enzyme lipoamide dehydrogenase (LADH) at physiological concentrations (Ka = 0.1 µM free zinc), inhibiting lipoamide and accelerating NADH dehydrogenase activities. These differential effects have been attributed to coordination of Zn2+ by LADH active-site cysteines. A detailed kinetic mechanism has now been developed for the diaphorase (NADH-dehydrogenase) reaction catalyzed by pig heart LADH using 2,6-dichlorophenol-indophenol (DCPIP) as a model quinone electron acceptor. Anaerobic stopped-flow experiments show that two-electron reduced LADH is 15-25-fold less active towards DCPIP reduction than four-electron reduced enzyme, or Zn2+-modified reduced LADH (the corresponding values of the rate constants are (6.5 ± 1.5) × 103 M–1·s–1, (9 ± 2) × 104 M–1·s–1, and (1.6 ± 0.5) × 105 M–1·s–1, respectively). Steady-state kinetic studies with different diaphorase substrates show that Zn2+ accelerates reaction rates exclusively for two-electron acceptors (duroquinone, DCPIP), but not for one-electron acceptors (benzoquinone, ubiquinone, ferricyanide). This implies that the two-electron reduced form of LADH, prevalent at low NADH levels, is a poor two-electron donor compared to the four-electron reduced or Zn2+-modified reduced LADH forms. These data suggest that zinc binding to the active-site thiols switches the enzyme from one- to two-electron donor mode. This zinc-activated switch has the potential to alter the ratio of superoxide and H2O2 generated by the LADH oxidase activity.
KEY WORDS: ubiquinone, 2,6-dichlorophenol indophenol, duroquinone, ferricyanide, enzyme kinetics, dehydrolipoyl dehydrogenase

DOI: 10.1134/S0006297920080064